Proteins are made up of a string of sub-units called amino acids, the sequence of which is known as the primary structure of the protein. This first level of organisation of the protein is directed by the gene sequence encoding the protein, wherein a sequence of three nucleic acids (a codon) in the gene specifies the nature of the amino acid at any particular position. In addition to the primary structure, most proteins also exhibit a higher level of structural organisation. It is this three dimensional, or tertiary, structure, which allows the protein to function in its biological role. Many proteins in the cell exist as aggregates of two or more folded proteins, or sub-units. This level of organisation is referred to as the quaternary structure of a protein.
Typically, proteins are made up of a number of folded domains, i.e. compact regions of folded structure. Several varieties of domains exist, including α-helices, β-sheets and β-turns.
A folded protein is held in its secondary or tertiary structure by several types of bonds. These include electrostatic interactions, which occur between the oppositely charged side chains of the amino acids making up the primary structure; hydrogen bonds between amino acids; weak interactions between uncharged groups (known as van der Waals interactions): and disulphide bonds between cysteine amino acid residues. Unfolding a protein by reversing these interactions is known as denaturing the protein, back to its primary structure. This may be achieved by placing the protein in a high temperature environment, or in SDS solution.
The folding of a protein to its final, functional conformation is one of the last steps in protein production. It is a vital step in a complex process, and any error in the process can induce massive physiological problems. For example, there is strong evidence to suggest that Bovine Spongiform Encephalopathy (BSE) stems from a mis-folded protein (Horwich et al Cell 89 499-510 (1997)). An understanding of protein folding and stability will provide a clearer insight into the causes of disease, and therefore will allow the development of better treatments or preventative measures for disease.
There exist a number of techniques to study proteins. These include X-ray crystallography ((Blundell et al Protein Crystallography London: Academic press (1976)); NMR (Clore et al Progress in NMR Spectroscopy 23 43-92 (1991)); differential scanning calirometry (Blaber et al protein structure and Stability Florida State University (1995)); and unnatural amino acid engineering (Mendel et al Science 256 (5065) 1798-1802 (1992)).
X-ray crystallography is a preferred method for determining the three dimensional structure of proteins. However, this technique has the fundamental problem that it can only be employed when the proteins are crystallised, and this is not always easy or even possible. This puts constraints on the ability to study conformational changes in the protein, changes in the folding in response to changes in environment, or interaction with other factors.
Florescence and absorption by certain optically active amino acids in a protein have also been used to monitor conformational changes in protein and to measure protein concentration (Chen et al Biochemistry 37 9976-9982 (1998)). These amino acids contain an indole chromophore whose transitional geometry is responsible for the optical activity of the amino acid (Callis et al Chemical Physics Letters 244 53-58 (1995); Fender et al Chemical Physics Letters 262 343-348 (1996) and Fender et al Chemical Physics Letters 239 31-37 (1995)). The main amino acids contributing to this are tryptophan and tyrosine, while phenylalanine and the disulphide bond between cysteine residues also show some fluorescence.
The problem encountered with all of the above mentioned techniques is that they are not capable of real-time protein folding analysis. This imposes significant constraints on protein folding and stability analysis, and therefore also on the use of proteins in the diagnosis and prevention and/or treatment of disease. Furthermore, current systems often reveal little about the relationship between the dynamics of the folding and the static conformational information available.
The present invention will be useful in any method where it is desirable to analyse the folding and/or stability of a protein, and/or the interaction of a protein with biological factors including ligands, receptors, sugars, hormones, nucleic acids and therapeutics agents. Other long chain or macromolecules may also be studied, as long as they are able to fold or otherwise change their physical configuration in response to a temperature change.